RF9 powerfully stimulates gonadotrophin secretion in the ewe: evidence for a seasonal threshold of sensitivity.

GPR147 and its endogenous ligands, RFRPs, are emerging as important actors in hypothalamic-pituitary axis control. The role of this system would be to inhibit gonadotrophin secretion. However, data on the subject are contradictory. The discovery of RF9 (adamantanecarbonyl-RF-2-NH(2)), a GPR147 antagonist, prompted us to use this new tool to further investigate this system in the ewe. Accordingly, we tested the effect of i.c.v. administration of RF9 on gonadotrophin secretion in the ewe during anoestrous and the breeding season. Intracerebroventricular injections of RF9 (from 50-450 nmol) caused a clear elevation in peripheral blood plasma luteinising hormone (LH) concentrations. The effect of RF9 on LH was more pronounced during the anoestrous season. Furthermore, peripheral administration of RF9 as a bolus (2.1, 6.2 and 12.4 µmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6 µmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4 µmol bolus i.v.) has an effective half life of 5.5 h in the plasma. Conversely, RF9 is not detectable in the cerebrospinal fluid, suggesting that it does not cross the blood-brain barrier. The increase in LH plasma concentrations induced by RF9 was blocked by previous administration of 1.3 µmol per ewe of gondotrophin-releasing hormone (GnRH) antagonist Teverelix. This suggests that GnRH is involved in the stimulatory effect of RF9 on gonadotrophin secretion. Finally, no variation in LH plasma concentrations could be detected in ovariectomised ewes injected either i.c.v. or i.v. with RFRP3 (VPNLPQRF-NH(2)). The lack of effect of RFRP3 in our experimental setting suggests that the mechanisms involved in RF9 action are probably more complex than previously assumed. Our results indicate that delivery of RF9 in the ewe greatly increases gondadotrophin secretion in both the oestrus and anoestrus season, suggesting a potential new way of controlling reproduction in mammals.

Characterization of the inhibitory roles of RFRP3, the mammalian ortholog of GnIH, in the control of gonadotropin secretion in the rat: in vivo and in vitro studies.

RF-amide related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone (GnIH), have been proposed as key regulators of gonadotropin secretion in higher vertebrates. Yet considerable debate has arisen recently on their physiological relevance and potential mechanisms and sites of action. Present studies were undertaken to further characterize the effects of RFRP on LH and FSH secretion by a combination of in vivo and in vitro approaches in male and female rats. Initial screening via intracerebroventricular (icv) administration of different analogs of RFRP1 (RFRP1-12 and RFRP1-20) and RFRP3 (RFRP3-8 and RFRP3-17), as well as the related neuropeptide FF (NPFF8), to gonadectomized (GNX) female rats evidenced significant, albeit modest, inhibitory effects on LH secretion only for RFRP3-8 and RFRP3-17, which were detectable at the high dose rage (1 nmol for RFRP3-8, 5 nmol for RFRP3-17). This moderate inhibitory action was also documented after icv administration of RFRP3-8 to intact and GNX male rats. In addition, systemic (intravenous) administration of RFRP3-8 decreased the circulating levels of both gonadotropins in GNX male rats. Likewise, RFRP3-8 inhibited basal and GnRH-stimulated LH secretion by pituitaries from GNX males in vitro. This inhibitory effect was blocked by the antagonist of RFRP receptors, RF9. In summary, our results support a putative inhibitory role of RFRP3 as ortholog of GnIH in the regulation of gonadotropin secretion in mammals, which appears to involve direct pituitary actions as well as potential central (hypothalamic) effects.

Characterization of the potent gonadotropin-releasing activity of RF9, a selective antagonist of RF-amide-related peptides and neuropeptide FF receptors: physiological and pharmacological implications.

Identification of RF-amide-related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone, has drawn considerable interest on its potential effects and mechanisms of action in the control of gonadotropin secretion in higher vertebrates. Yet, these analyses have so far relied mostly on indirect approaches, while direct assessment of their physiological roles has been hampered by the lack of suitable antagonists. RF9 was recently reported as a selective and potent antagonist of the receptors for RFRP (RFRPR) and the related neuropeptides, neuropeptide FF (NPFF) and neuropeptide AF (NPFF receptor). We show here that RF9 possesses very strong gonadotropin-releasing activities in vivo. Central administration of RF9 evoked a dose-dependent increase of LH and FSH levels in adult male and female rats. Similarly, male and female mice responded to intracerebroventricular injection of RF9 with robust LH secretory bursts. In rats, administration of RF9 further augmented the gonadotropin-releasing effects of kisspeptin, and its stimulatory effects were detected despite the prevailing suppression of gonadotropin secretion by testosterone or estradiol. In fact, blockade of estrogen receptor-alpha partially attenuated gonadotropin responses to RF9. Finally, systemic administration of RF9 modestly stimulated LH secretion in vivo, although no direct effects in terms of gonadotropin secretion were detected at the pituitary in vitro. Altogether, these data are the first to disclose the potent gonadotropin-releasing activity of RF9, a selective antagonist of RFRP (and NPFF) receptors. Our findings support a putative role of the RFRP/gonadotropin-inhibitory hormone system in the central control of gonadotropin secretion in mammals and have interesting implications concerning the potential therapeutic indications and pharmacological effects of RF9.

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp(90) residue.

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Three distinct types of GnRH receptor characterized in the bullfrog.

It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.

Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization.

The gonadotropin-releasing hormone receptor (GnRH-R) of the African catfish couples to phospholipase C and belongs to the large family of G protein-coupled receptors. We recently demonstrated that removal of the carboxyl-terminal tail (S331-Q379) from the catfish GnRH-R results in a loss of agonist binding; the current study sought to define more precisely the role of this region in receptor function. Progressive truncations of the carboxyl-terminal tail decreased cell surface expression detected by either enzyme-linked immunosorbent assay or agonist-binding. The two most truncated receptors (stop331 and stop337) showed no binding but were detected at the cell surface by enzyme-linked immunosorbent assay. All receptors able to bind agonist were also able to activate phospholipase C. The catfish GnRH-R was phosphorylated after agonist-occupation and use of truncated mutants showed this phosphorylation to be within the carboxyl-terminal tail. Furthermore, studies with S356A, S363A and SS356,363AA mutant receptors demonstrated that Ser363 is a major site of agonist-induced phosphorylation. The absence of this phospho-acceptor site markedly impaired agonist-mediated receptor internalization. In addition, both, Ser363 and the last 12 residues of the tail (not containing Ser363) were shown to be important for beta-arrestin-dependent internalization. These observations are relevant to the regulatory function of the carboxyl-terminal tail of G protein-coupled receptors in general and are particularly intriguing given the absence of this region in mammalian GnRH-Rs.

Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization.

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is, at present, the only G-protein-coupled receptor that activates phospholipase C and lacks a C-terminal tail. We have previously demonstrated that this unique structural feature is associated with resistance to rapid desensitization of phosphoinositide signaling in COS-7 and HEK-293 cells (Heding, A., Vrecl, M., Bogerd, J., McGregor, A., Sellar, R., Taylor, P. L., and Eidne, K. A. (1998) J. Biol. Chem. 273, 11472-11477). Using receptors tagged with a nonapeptide of the influenza hemagglutinin protein to enable immunoprecipitation, we now demonstrate that the mammalian GnRH-R is not phosphorylated in an agonist-dependent manner. In contrast, the mammalian thyrotropin-releasing hormone receptor and the African catfish GnRH-R, both of which have a C-terminal tail, are phosphorylated in response to agonist challenge. Furthermore, chimeras of the mammalian GnRH-R with the C-terminal tail of either the mammalian thyrotropin-releasing hormone receptor or the catfish GnRH-R are also phosphorylated in an agonist-dependent manner. Only those receptors having C-terminal tails showed desensitization of phosphoinositide responses within 5-10 min of agonist challenge. We also show that the internalization of all these receptors when expressed transiently in COS-7 cells is similar. This dissociates receptor internalization from rapid desensitization and demonstrates that the lack of a C-terminal tail in the mammalian GnRH-R results in an inability of the receptor to undergo agonist-dependent phosphorylation and that this results directly in a resistance to rapid desensitization.

Characterization of neocortical and hippocampal synaptosomes from temporal lobe epilepsy patients.

To investigate epilepsy-associated changes in the presynaptic terminal, we isolated and characterized synaptosomes from biopsies resected during surgical treatment of drug-resistant temporal lobe epilepsy (TLE) patients. Our main findings are: (1) The yield of synaptosomal protein from biopsies of epilepsy patients was about 25% of that from rat brain. Synaptosomal preparations were essentially free of glial contaminations. (2) Synaptosomes from TLE patients and naive rat brain, quickly responded to K(+)-depolarization with a 70% increase in intrasynaptosomal Ca(2+) ([Ca(2+)](i)), and a 40% increase in B-50/GAP-43 phosphorylation. (3) Neocortical and hippocampal synaptosomes from TLE patients contained 20-50% of the glutamate and gamma-aminobutyric acid (GABA) contents of rat cortical synaptosomes. (4) Although the absolute amount of glutamate and GABA released under basal conditions from neocortical synaptosomes of TLE patients was lower than from rat synaptosomes, basal release expressed as percentage of total content was higher (16.4% and 17.3%, respectively) than in rat (11.5% and 9. 9%, respectively). (5) Depolarization-induced glutamate and GABA release from neocortical synaptosomes from TLE patients was smaller than from rat synaptosomes (3.9% and 13.0% vs. 21.9% and 25.0%, respectively). (6) Analysis of breakdown of glial fibrillary acid protein (GFAP) indicates that resection time (anoxic period during the operation) is a critical parameter for the quality of the synaptosomes. We conclude that highly pure and viable synaptosomes can be isolated even from highly sclerotic human epileptic tissue. Our data show that in studies on human synaptosomes it is of critical importance to distinguish methodological (i.e., resection time) from pathology-related abnormalities.

Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

Differences in structure-function relations between nonmammalian and mammalian gonadotropin-releasing hormone receptors.

Mammalian gonadotropin-releasing hormone receptors (GnRH-Rs) differ from other G protein-coupled receptors in lacking the intracellular C-terminus and in showing an exchange of two otherwise highly conserved Asp (D) and Asn (N) residues in transmembrane domains (TMD) 2 and 7, respectively. However, the first GnRH-R characterized from a nonmammalian vertebrate, the African catfish, does contain an intracellular C-terminus and has D residues in TMD 2 and 7. The functional relevance of these structural features was analysed with D90N321, N90D321, N90N321 and C-terminally truncated mutant catfish GnRH-Rs. An antiserum raised against the recombinant extracellular domain of the wild-type catfish GnRH-R detected all mutant receptors at the cell surface of transiently transfected 293T cells. However, only the D90N321 mutant specifically bound GnRHs and activated signal transduction in response to GnRHs; all other mutants were inactive in both respects. We conclude that the catfish GnRH-R differs from the mammalian GnRH-Rs in that both the C-terminal domain and D90 in TMD 2 are important for receptor functioning.

Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor.

A cDNA encoding a putative gonadoliberin receptor was cloned from the pituitary of the African catfish. Conceptual translation predicts a protein of 379 amino acids which shows typical characteristics of GTP-binding-protein-coupled receptors. The isolated cDNA was stable expressed in human embryonic kidney (HEK) 293 cells which were used for studies on gonadoliberin-activated second messenger systems (inositol phosphate production; increase in cAMP and/or intracellular Ca2+). The isolated cDNA encoded a functional receptor, designated catfish gonadoliberin receptor (cfGnRH-R), which had an amino acid sequence similarity of 38% with mammalian gonadoliberin receptors. In contrast to its mammalian counterparts which lack an intracellular carboxy-terminal domain, the cfGnRH-R contains an additional 49 amino acid residues. From the two endogenous gonadoliberins in African catfish, chicken gonadoliberin-II had a several hundredfold higher potency than catfish gonadoliberin to activate cfGnRH-R-associated second messenger systems in transfected HEK 293 cells. This is in line with the previously determined higher gonadotropin-release capacity of chicken gonadoliberin-II in catfish. Stimulation of second messenger systems with chicken gonadoliberin-II, but not with catfish gonadoliberin, resulted in a biphasic effect and chicken gonadoliberin-II led to a higher maximum stimulation than catfish gonadoliberin. Challenging cfGnRH-R simultaneously with chicken gonadoliberin-II and catfish gonadoliberin did not lead to additive effects. In contrast, two types of mutual inhibitory effects were recorded. These data indicate that a single cognate cfGnRH-R couples with distinct efficacies to signal transduction systems upon stimulation by the two endogenous gonadoliberins which, in addition, may interact negatively.